Formation and Function of Human CD28-negative T cells. A central problem in immunology is how Tcell memory is maintained without antigen. CD28-negative "knockout" mice initiate immune responses but have reduced T cell memory. It is unclear how CD28 is required for initiation and/or maintenance of memory. CD28 co-stimulates T cell responses to antigen, but also stimulates a subset of T cells without antigen ("CD28agonism"). In aging, chronic infection and autoimmunity, up to 75% of human but not mouse CD8+ T cells are CD28-negative. These do not proliferate, associated with immune deficits, including failure to contain HIV. Lack of CD28 may explain proliferative failure of CD28-negative CD8+ T cells, but inhibitory NK receptors onCD28-negative cells might also inhibit proliferation. We hypothesize that CD28 co-stimulates recall responses but also maintains memory T cells without antigen. CD28 agonism is triggered by B7.1 (CDS0) on antigen-presenting cells and by certain monoclonal antibodies. These agonists trigger tyrosine phosphorylation ofCD28, recruit PI3 kinase, sustain expression of CD25 and the NK receptor-family member CD69 and result in T cell proliferation or apoptosis. CD28 agonism also suppresses CD28 transcription. Therefore, CD28agonism might sustain CD28+ memory cells without antigen, but might also drive the formation of CD28-negative T cells. The long-range goal is to understand how expression of and signaling by CD28 contributes toT cell memory. The Specific Aims are (1) identify genes and T cell subsets responding to CD28 agonism(2) identify the CD28 isoforms and proximal mechanisms for CD28 agonism; (3) construct mice expressingfloxed-huCD28 to permit controlled deletion of CD28 expression in a mouse model. These studies are important for understanding the biology of human memory T cells especially for vaccine design.